Determining the role of CLN3 in the eye

确定 CLN3 在眼睛中的作用

基本信息

批准号：
10357934
负责人：
Ruchira Singh
金额：
$ 45.82万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10357934
关键词：
Affect Animal Model Apical Atrophic Binding Blindness CLN3 gene Cadaver Cell Death Cell Fractionation Cell physiology Cell surface Cells Cellular Structures Chronic Clinical Data Dextrans Digestion Disease Epithelial Epithelial Physiology Evolution Excision Eye Family suidae Functional disorder Genotype Health Homeostasis Human Image Immunohistochemistry Impairment Ingestion Knowledge Laboratories Lead Light Link Lipofuscin Longitudinal Studies Mediating Molecular Mus Mutation Nerve Degeneration Nutrient Pathology Patient imaging Patients Pattern Phagocytosis Pharmacotherapy Phenotype Photoreceptors Play Proteins Recycling Retina Retinal Degeneration Retinal Photoreceptors Rodent Role Secondary to Structure Structure of retinal pigment epithelium Techniques Testing Transportation Vision Vitamin A Waste Products cellular imaging cellular microvillus disease-causing mutation early childhood experimental study feeding gene correction histological studies imaging study in vivo induced pluripotent stem cell insight internal control juvenile neuronal ceroid lipofuscinosis mouse model multimodality novel porcine model retinal imaging retinal progenitor cell uptake visual cycle

项目摘要

ABSTRACT Juvenile neuronal ceroid lipofuscinosis (JNCL), caused by mutations in the CLN3 gene, leads to neurodegeneration and vision loss in early childhood. Vision loss in JNCL is due to retinal degeneration. Specifically, in the retina, JNCL affects both the light sensing photoreceptor cells and their underlying epithelium, RPE. Furthermore, photoreceptor cell loss in the JNCL retina has been linked to vision loss. However, the molecular mechanism(s) underlying photoreceptor cell loss in JNCL are currently not known. Preliminary studies in our laboratory utilizing human induced pluripotent stem cell (hiPSC)-derived retinal cells suggest a novel role of CLN3 in maintaining key RPE structure (microvilli) and function (uptake of shed photoreceptor outer segments or POS) that are essential for photoreceptor survival and therefore vision. Specifically, JNCL hiPSC-RPE display microvilli disorganization/ballooning and impaired phagocytosis of POS compared to control hiPSC-RPE cells. Notably, consistent with RPE microvilli dysfunction and reduced POS uptake in JNCL hiPSC-RPE cells, RPE in the eyes of JNCL patients have reduced lipofuscin (breakdown products of POS digestion). Together, these data suggest that “CLN3 is a RPE microvilli resident protein that regulates POS uptake and CLN3 dysfunction in JNCL leads to impaired POS phagocytosis and subsequently decreased lipofuscin accumulation in JNCL RPE cells.” To test this hypothesis, in this project, we propose to employ ultrastructural and molecular studies on control and JNCL hiPSC-RPE cells. Specifically, we will first utilize immunohistochemistry, RNAScope and subcellular fractionation techniques to confirm the localization of a proportion of CLN3 in RPE cells to the apical microvilli in primary (mouse, porcine, human) and control hiPSC- RPE. Next, we will compare i) microvilli structure and molecular composition and ii) POS binding and internalization by control vs. JNCL hiPSC-RPE cells. Furthermore, to correlate the reduced POS phagocytosis by JNCL hiPSC-RPE cells to reduced lipofuscin (autofluorescence accumulation) seen in JNCL RPE in vivo, we will evaluate the pattern of autofluorescence accumulation (cell surface vs. intracellular) in parallel cultures of control and JNCL hiPSC-RPE after chronic POS feeding (1-3 months). We will also validate our findings on patient hiPSC-RPE cells using primary RPE/POS from a JNCL mouse and porcine model. Finally, we will utilize in vivo retinal imaging of patients with JNCL in a longitudinal study to document the presence and amount of autofluorescence/lipofuscin in the photoreceptor-RPE cell layer in relationship to photoreceptor vs. RPE cell loss in patients progressing through the disease. Altogether, the knowledge gained from this study will provide novel insights into i) the role of CLN3 in RPE physiology and ii) the plausible contribution of RPE dysfunction to JNCL retinal pathophysiology. !

抽象的由 CLN3 基因突变引起的幼年神经元蜡样质脂褐质沉积症 (JNCL) JNCL 儿童早期的神经变性和视力丧失是由视网膜变性引起的。具体来说，在视网膜中，JNCL 影响感光感光细胞及其底层细胞。此外，JNCL 视网膜中的感光细胞损失与视力丧失有关。然而，JNCL 中感光细胞损失的分子机制目前尚不清楚。我们实验室利用人类诱导多能干细胞 (hiPSC) 来源的视网膜细胞进行的初步研究表明 CLN3 在维持关键 RPE 结构（微绒毛）和功能（脱落细胞的吸收）方面具有新作用。光感受器外节（POS）对于光感受器的生存和视力至关重要。具体来说，JNCL hiPSC-RPE 显示微绒毛紊乱/膨胀和 POS 吞噬作用受损与对照 hiPSC-RPE 细胞相比，值得注意的是，与 RPE 微绒毛功能障碍和 POS 减少一致。 JNCL hiPSC-RPE细胞摄取，JNCL患者眼中的RPE脂褐素减少（分解 POS 消化的产物），这些数据共同表明“CLN3 是一种 RPE 微绒毛驻留蛋白，调节 POS 摄取，JNCL 中 CLN3 功能障碍导致 POS 吞噬作用受损，随后 JNCL RPE 细胞中脂褐素的积累。”为了检验这一假设，在本项目中，我们建议减少至具体来说，我们将首先对对照细胞和 JNCL hiPSC-RPE 细胞进行超微结构和分子研究。利用免疫组织化学、RNAScope 和亚细胞分级分离技术来确认 RPE 细胞中 CLN3 与原代（小鼠、猪、人）和对照 hiPSC 中顶端微绒毛的比例接下来，我们将比较 i) 微绒毛结构和分子组成以及 ii) POS 结合和对照与 JNCL hiPSC-RPE 细胞的内化此外，将 POS 吞噬作用的减少关联起来。 JNCL hiPSC-RPE 细胞减少体内 JNCL RPE 中观察到的脂褐素（自发荧光积累），我们将评估平行培养物中自发荧光积累的模式（细胞表面与细胞内）我们还将验证我们的研究结果。最后，我们将利用来自 JNCL 小鼠和猪模型的原代 RPE/POS 患者 hiPSC-RPE 细胞。在一项纵向研究中对 JNCL 患者进行体内视网膜成像，记录 JNCL 的存在和数量光感受器-RPE 细胞层中的自发荧光/脂褐质与光感受器与 RPE 细胞的关系总而言之，从这项研究中获得的知识将提供疾病进展中患者的损失。对 i) CLN3 在 RPE 生理学中的作用和 ii) RPE 功能障碍的可能贡献的新见解 JNCL 视网膜病理生理学。！